State the application's broad long-term objectives and specific aims, making reference to the health relatedness of the project. Describe concisely the research design and methods for achieving these goals. Avoid summaries of past accomplishments and the use of the first person. This description is meant to serve as a succinct and accurate description of the proposed work when separated from the application. If the application is funded, this During the present cycle of funding we found that autocrine TGFbeta was induced by vitamin D3 analogs in ER+ breast cancer and pre-malignant mammary epithelial cells. Vitamin D3 analog response was 100 fold more resistant to inhibition in the absence of autocrine TGFbeta and preliminary evidence suggests that Smad3 activated by TGFbeta signaling enhances vitamin D3 receptor VDR directed transcription. However, while transfection of the type II TGFbeta receptor (RII) into RII null MCF-7 cells completely restores vitamin D analog sensitivity, transfection of Smad3 does not. This indicates that a second aspect of TGFbeta signaling is required in addition to Smad3. Thus, in Specific Aim I we will test the hypothesis that Smad3 interactions with the VDR are required for TGFbeta/VDR crosstalk, but that in addition to this Smad3 pathway, TGFbeta mediated activation of the MAPK pathway is also required. We and others have observed that vitamin D analogs cause apoptosis. The mechanism by which apoptosis is induced is largely unexplored, particularly with respect to the role of TGFbeta crosstalk. TGFbeta itself also induces apoptosis. Consequently, we will test the hypothesis that vitamin D3 analog induced apoptosis is mediated by the enhanced autocrine TGFbeta activity resulting from treatment with the drug. During the present period of funding we found that TGFbeta receptor expression in ER+ breast cancer cells is transcriptionally repressed. The repression can be blocked by treatment with the methylase inhibitor, 5 aza deoxycytidine. Surprisingly, this agent did not reverse methylation of the RII promoter, but instead increased the cellular level of Sp1 which is underexpressed in ER+ breast cancer cells. Sp1 is required for RII transcription. Interestingly, Sp1 transcription was not targeted by 5 aza deoxycytidine. Further, investigation showed that Sp3 transcription factor which represses Sp1 mediated transcription was elevated in ER+ breast cancer cell lines. Sp3 was shown to be a repressor of RII by southwestern, gel shift and blockade of RII promoter-reporter activity after Sp3 transfection. In addition Sp3 transcripts were decreased as a result of 5 aza cytidine treatment. Consequently, it appears as though Sp1/Sp3 homeostasis is responsible for repression of RII transcription in ER+ breast cancer cells. This hypothesis will be tested in Specific Aim III. The Specific Aims for the renewal project are: 1. Determine the mechanism of crosstalk between vitamin D3 analogs and TGFbeta in ER+ breast cancer cells, immortalized mammary epithelial cells and normal mammary epithelial cells. Determine whether vitamin D3 response in ER- cells is also associated with TGFbeta crosstalk. II. Determine the mechanism of induction of apoptosis by vitamin D3 analogs in the cell types listed in Specific Aim I and determine whether apoptosis inhibition is dependent upon autocrine TGFbeta. III. Determine how Sp1 and Sp3 homeostasis is controlled in ER+ breast cancer to regulate RII transcription.